Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase
| Autor: | F. L. Crane, L. M. McCann, Graeme B. Cox, Frank Gibson, J. D. Butlin |
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| Jazyk: | angličtina |
| Rok vydání: | 1973 |
| Předmět: |
History
Mutant chemistry.chemical_element Ultrafiltration Calcium Bioenergetics medicine.disease_cause Dithiothreitol Education chemistry.chemical_compound Adenosine Triphosphate Polysaccharides Genes Regulator medicine Escherichia coli Magnesium Magnesium ion Adenosine Triphosphatases Chromatography Strain (chemistry) Chemistry Cell Membrane Molecular biology Computer Science Applications Enzyme Activation Kinetics Membrane Biochemistry Solubility Mutation Chromatography Gel Triphosphatase Spectrophotometry Ultraviolet Oxidoreductases Ultracentrifugation |
| Popis: | 1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 2. The Mg(2+),Ca(2+)-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA(-)) could not be re-activated by the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA(-)). |
| Databáze: | OpenAIRE |
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